A patch clamp set-up and thermocycler for polymerase chain reaction: tools for understanding neuronal excitability and gene expression

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Animals must consume energy to balance expenditures, but what are the signals that tell animals when to eat?  How are these signals communicated to the brain?  How do neurons respond to these signals?  These questions are central to understanding sensation and perception of hunger, and regulation of energy homeostasis.      A group of neurons in the brain, called subfornical organ (SFO) neurons are specialized to detect levels of hormones circulating in the blood, including hormones that regulate hunger and feeding (such as leptin and insulin).  My preliminary experiments indicate that rat SFO neurons exhibit changes in gene expression after a 48 hour fast.  Two of the down-regulated genes encode voltage gated Na+ channels, proteins known to directly control neuronal electrical excitability.  The goal of this proposal is to understand physiological roles of observed changes in SFO neuron gene expression.  This proposal outlines three specific objectives.  The first objective is to identify which population of neurons in the rat SFO is altered by a 48h fast, and to describe in detail changes in the electrical properties of these neurons.  The second objective is to investigate whether changes in hormones that regulate hunger and feeding may be responsible for driving the observed changes in gene expression and electrical properties.  The third objective is to determine the specific contributions of one of the down-regulated Na+ channel proteins to the regulation of electrical activity of SFO neurons.  These objectives will be accomplished using a combination of methods including immunohistochemistry, patch clamp electrophysiological recording, reverse transcription coupled polymerase chain reaction (RT-PCR), real time PCR and siRNA mediated inhibition of gene expression.        This proposal is requesting funds for the purchase of electrophysiology equipment and a thermocycler (for RTPCR) that are essential to carry out this research program.  Together, this research will increase our understanding of the molecular physiology of how the central nervous system percieves hunger signals and regulates energy balance.